AML with complex karyotype: extreme genomic complexity revealed by combined long-read sequencing and Hi-C technology.

Acute myeloid leukemia with complex karyotype (CK-AML) is associated with poor prognosis, which is only in part explained by underlying TP53 mutations. Especially in the presence of complex chromosomal rearrangements, such as chromothripsis, the outcome of CK-AML is dismal. However, this degree of complexity of genomic rearrangements contributes to the leukemogenic phenotype and treatment resistance of CK-AML remains largely unknown. Applying an integrative workflow for the detection of structural variants (SVs) based on Oxford Nanopore (ONT) genomic DNA long-read sequencing (gDNA-LRS) and high-throughput chromosome confirmation capture (Hi-C) in a well-defined cohort of CK-AML identified regions with an extreme density of SVs. These rearrangements consisted to a large degree of focal amplifications enriched in the proximity of mammalian-wide interspersed repeat elements, which often result in oncogenic fusion transcripts, such as USP7::MVD, or the deregulation of oncogenic driver genes as confirmed by RNA-seq and ONT direct complementary DNA sequencing. We termed this novel phenomenon chromocataclysm. Thus, our integrative SV detection workflow combing gDNA-LRS and Hi-C enables to unravel complex genomic rearrangements at a very high resolution in regions hard to analyze by conventional sequencing technology, thereby providing an important tool to identify novel important drivers underlying cancer with complex karyotypic changes.

Pediatric Extracranial Germ Cell Tumors: Expression of microRNA.

BACKGROUND: Germ cell tumors (GCTs) may occur from the neonatal period to late adulthood, characterized by extensive clinical and pathologic heterogeneity. MicroRNAs are a family of small noncoding RNAs that regulate a wide array of biological processes including carcinogenesis. MicroRNAs may be used for many purposes in clinical diagnostics. Numerous studies have proven the diagnostic value of microRNA371-373 and microRNA302/367 expression in malignant GCT. The diagnostic value of microRNA375 is disputable, because while its value is confirmed by some research data, there are still others denying it. METHODS: The results of our own research on the relative expression of 10 microRNAs, including microRNA375, associated with GCT in the tumor tissues of 84 children and adolescents are presented. RESULTS: In our research, overexpression of microRNA 371-373, 302/367 detected in the group of malignant GCT subtypes. Statistically significant expression of microRNA375 have been defined not only in the group of malignant GCT subtypes, but also in the group of immature teratomas. Among malignant GCTs, high expression of microRNA375 is specific for yolk sac tumors. In the group of seminomas, embryonic carcinomas, and mature teratomas expression of microRNA375 was observed imperceptible, even so the results were statistically insignificant. CONCLUSION: Expression of microRNA 371-373, 302/367 is representative of malignant GCT subtypes. Statistically significant and high expression of microRNA375 attributable for yolk sac tumors and immature teratomas.

Relapse of NPM1-Mutated AML with Extramedullary Manifestation 17 Years after Allogeneic Hematopoietic Stem Cell Transplantation.

The majority of patients with acute myeloid leukemia (AML) with the NPM1 mutation achieve remission with intensive chemotherapy. However, many patients subsequently relapse, which occurs frequently within the first 2-3 years after therapy, while late relapse after more than 10 years is rare and can also represent secondary/therapy-associated AML without the NPM1 mutation. Here, we present a case of NPM1-mutated AML that developed medullary and extramedullary relapse 17 years after allogeneic stem cell transplantation, maintaining the NPM1 mutation and all other genetic alterations detected at first diagnosis. This exceptionally long latency between diagnosis and relapse of a genetically highly related leukemic clone implies the existence of therapy-resistant, persisting dormant leukemic stem cells in NPM1 mutant AML.

Polymorphisms in CXCR3 ligands predict early CXCL9 recovery and severe chronic GVHD.

Chronic graft-versus-host disease (cGVHD) is a major cause of mortality and morbidity after allogeneic stem cell transplantation (alloSCT). The individual risk of severe cGVHD remains difficult to predict and may involve CXCR3 ligands. This study investigated the role of single-nucleotide polymorphisms (SNPs) of CXCL4, CXCL9, CXCL10, and CXCL11, and their day +28 serum levels, in cGVHD pathogenesis. Eighteen CXCR3 and CXCL4, CXCL9-11 SNPs as well as peri-transplant CXCL9-11 serum levels were analyzed in 688 patients without (training cohort; n = 287) or with statin-based endothelial protection cohort (n = 401). Clinical outcomes were correlated to serum levels and SNP status. Significant polymorphisms were further analyzed by luciferase reporter assays. Findings were validated in an independent cohort (n = 202). A combined genetic risk comprising four CXCR3 ligand SNPs was significantly associated with increased risk of severe cGVHD in both training cohort (hazard ratio (HR) 2.48, 95% confidence interval (CI) 1.33-4.64, P = 0.004) and validation cohort (HR 2.95, 95% CI 1.56-5.58, P = 0.001). In reporter assays, significantly reduced suppressive effects of calcineurin inhibitors in constructs with variant alleles of rs884304 (P < 0.001) and rs884004 (P < 0.001) were observed. CXCL9 serum levels at day +28 after alloSCT correlated with both genetic risk and risk of severe cGVHD (HR 1.38, 95% CI 1.10-1.73, P = 0.006). This study identifies patients with high genetic risk to develop severe cGVHD.

Long-Term Signs of T Cell and Myeloid Cell Activation After Intestinal Transplantation With Cellular Rejections Contributing to Further Increase of CD16+ Cell Subsets.

The intestine mediates a delicate balance between tolerogenic and inflammatory immune responses. The continuous pathogen encounter might also augment immune cell responses contributing to complications observed upon intestinal transplantation (ITx). We thus hypothesized that ITx patients show persistent signs of immune cell activation affecting both the adaptive and innate immune cell compartment. Information on the impact of intestinal grafts on immune cell composition, however, especially in the long-term is sparse. We here assessed activated and differentiated adaptive and innate immune subsets according to time, previous experience of cellular or antibody-mediated rejections or type of transplant after ITx applying multi-parametric flow cytometry, gene expression, serum cytokine and chemokine profiling. ITx patients showed an increase in CD16 expressing monocytes and myeloid dendritic cells (DCs) compared to healthy controls. This was even detectable in patients who were transplanted more than 10 years ago. Also, conventional CD4+ and CD8+ T cells showed persistent signs of activation counterbalanced by increased activated CCR4+ regulatory T cells. Patients with previous cellular rejections had even higher proportions of CD16+ monocytes and DCs, whereas transplanting higher donor mass with multi-visceral grafts was associated with increased T cell activation. The persistent inflammation and innate immune cell activation might contribute to unsatisfactory results after ITx.

Genomic landscape and clonal evolution of acute myeloid leukemia with t(8;21): an international study on 331 patients.

Acute myeloid leukemia with t(8;21)(q22;q22) is characterized by considerable clinical and biological heterogeneity leading to relapse in up to 40% of patients. We sequenced coding regions or hotspot areas of 66 recurrently mutated genes in a cohort of 331 t(8;21) patients. At least 1 mutation, in addition to t(8;21), was identified in 95%, with a mean of 2.2 driver mutations per patient. Recurrent mutations occurred in genes related to RAS/RTK signaling (63.4%), epigenetic regulators (45%), cohesin complex (13.6%), MYC signaling (10.3%), and the spliceosome (7.9%). Our study identified mutations in previously unappreciated genes: GIGYF2, DHX15, and G2E3 Based on high mutant levels, pairwise precedence, and stability at relapse, epigenetic regulator mutations were likely to occur before signaling mutations. In 34% of RAS/RTKmutated patients, we identified multiple mutations in the same pathway. Deep sequencing (∼42 000×) of 126 mutations in 62 complete remission samples from 56 patients identified 16 persisting mutations in 12 patients, of whom 5 lacked RUNX1-RUNX1T1 in quantitative polymerase chain reaction analysis. KIT high mutations defined by a mutant level ≥25% were associated with inferior relapse-free survival (hazard ratio, 1.96; 95% confidence interval, 1.22-3.15; P = .005). Together with age and white blood cell counts, JAK2, FLT3-internal tandem duplicationhigh, and KIT high mutations were identified as significant prognostic factors for overall survival in multivariate analysis. Whole-exome sequencing was performed on 19 paired diagnosis, remission, and relapse trios. Exome-wide analysis showed an average of 16 mutations with signs of substantial clonal evolution. Based on the resemblance of diagnosis and relapse pairs, genetically stable (n = 13) and unstable (n = 6) subgroups could be identified.

Role of Donor Clonal Hematopoiesis in Allogeneic Hematopoietic Stem-Cell Transplantation.

PURPOSE: Clonal hematopoiesis of indeterminate potential (CHIP) occurs in the blood of approximately 20% of older persons. CHIP is linked to an increased risk of hematologic malignancies and of all-cause mortality; thus, the eligibility of stem-cell donors with CHIP is questionable. We comprehensively investigated how donor CHIP affects outcome of allogeneic hematopoietic stem-cell transplantation (HSCT). METHODS: We collected blood samples from 500 healthy, related HSCT donors (age ≥ 55 years) at the time of stem-cell donation for targeted sequencing with a 66-gene panel. The effect of donor CHIP was assessed on recipient outcomes, including graft-versus-host disease (GVHD), cumulative incidence of relapse/progression (CIR/P), and overall survival (OS). RESULTS: A total of 92 clonal mutations with a median variant allele frequency of 5.9% were identified in 80 (16.0%) of 500 donors. CHIP prevalence was higher in donors related to patients with myeloid compared with lymphoid malignancies (19.2% v 6.3%; P ≤ .001). In recipients allografted with donor CHIP, we found a high cumulative incidence of chronic GVHD (cGVHD; hazard ratio [HR], 1.73; 95% CI, 1.21 to 2.49; P = .003) and lower CIR/P (univariate: HR, 0.62; 95% CI, 0.40 to 0.97; P = .027; multivariate: HR, 0.63; 95% CI, 0.41 to 0.98; P = .042) but no effect on nonrelapse mortality. Serial quantification of 25 mutations showed engraftment of 24 of 25 clones and disproportionate expansion in half of them. Donor-cell leukemia was observed in two recipients. OS was not affected by donor CHIP status (HR, 0.88; 95% CI, 0.65 to 1.321; P = .434). CONCLUSION: Allogeneic HSCT from donors with CHIP seems safe and results in similar survival in the setting of older, related donors. Future studies in younger and unrelated donors are warranted to extend these results. Confirmatory studies and mechanistic experiments are warranted to challenge the hypothesis that donor CHIP might foster cGVHD development and reduce relapse/progression risk.

Single Nucleotide Polymorphisms in CD40L Predict Endothelial Complications and Mortality After Allogeneic Stem-Cell Transplantation.

Purpose Endothelial vulnerability is a potential risk factor for complications after allogeneic stem-cell transplantation (alloSCT). The CD40/CD40 ligand (CD40L) axis contributes to inflammatory diseases and is upregulated in endothelial cells upon activation, suggesting a role in alloSCT biology. Here, we studied single nucleotide polymorphisms (SNPs) in the CD40L gene in recipients of alloSCT. Patients and Methods Three CD40L SNPs (rs3092920, rs3092952, rs3092936) were analyzed for association with transplant-associated thrombotic microangiopathy, overall nonrelapse mortality (NRM), and NRM after acute graft-versus-host disease in 294 recipients of alloSCT without statin-based endothelial prophylaxis (SEP). The significant genotype was then put into perspective with established thrombomodulin ( THBD) gene polymorphisms. Findings were validated in an independent cohort without SEP and in an additional 344 patients who received SEP. Results The rs3092936 CC/CT genotype was associated with an increased risk of transplant-associated thrombotic microangiopathy ( P = .001), overall NRM ( P = .03), and NRM after acute graft-versus-host disease ( P = .01). The rs3092936 CC/CT genotype was largely mutually exclusive of high-risk THBD SNPs. Both CD40L and THBD SNPs predicted adverse overall survival (OS) and overall NRM to a similar extent in training cohort (OS, P = .04; NRM, P < .001) and validation cohort (OS, P = .01; NRM, P = .001) without SEP. In contrast, SEP completely abolished the influence of the high-risk CD40L and THBD SNPs ( P = .40). Conclusion An increased risk of endothelial complications can be predicted before alloSCT by genetic markers in the recipient's genome. The normalization of mortality risks in patients treated with SEP suggests a way of overcoming the negative effect of high-risk genotypes and warrants further clinical validation.

Multiple myeloma cells modify VEGF/IL-6 levels and osteogenic potential of bone marrow stromal cells via Notch/miR-223.

Bone marrow mesenchymal stromal cells (BMMSCs) represent a crucial component of multiple myeloma (MM) microenvironment supporting its progression and proliferation. Recently, microRNAs have become an important point of interest for research on micro-environmental interactions in MM with some evidence of tumor supportive roles in MM. In this study, we examined the role of miR-223 for MM support in BMMSCs of 56 patients with MM (MM-BMMSCs). miR-223 expression in MM-BMMSCs was reduced by the presence of MM cells in vitro in a cell-contact dependent manner compared to mono-cultured MM-BMMSCs. Co-cultivation of MM cells and MM-BMMSCs induced activation of notch amongst others via jagged-2/notch-2 leading to increased expression of Hes1, Hey2, or Hes5 in both cell types. Cultivation of MM-BMMSCs with increasing levels of recombinant jagged-2 reduced miR-223 and increased Hes1 levels in a concentration-dependent manner. Transient reduction of miR-223 levels increased VEGF and IL-6 expression and secretion by MM-BMMSCs. In addition, reduction of miR-223 degraded the osteogenic differentiation potential of MM-BMMSCs. Inhibition of notch signaling induced apoptosis in both MM cells and MM-BMMSCs. Furthermore, it increased miR-223 levels and reduced expression of VEGF and IL-6 by both cell types. These data provide first evidence that miR-223 participates in different MM supporting pathways in MM-BMMSCs inlcuding regulation of cytokine secretion and expression as well as osteogenic differentiation of MM-BMMSCs. More insights on the role of miR-223 in MM-BMMSCs and in cellular interactions between MM cells and MM-BMMSCs could provide starting points for a more efficient anti-myeloma treatment by targeting of notch signaling. © 2015 Wiley Periodicals, Inc.

Profiling of somatic mutations in acute myeloid leukemia with FLT3-ITD at diagnosis and relapse.

Acute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive hematologic malignancy with a grave prognosis. To identify the mutational spectrum associated with relapse, whole-exome sequencing was performed on 13 matched diagnosis, relapse, and remission trios followed by targeted sequencing of 299 genes in 67 FLT3-ITD patients. The FLT3-ITD genome has an average of 13 mutations per sample, similar to other AML subtypes, which is a low mutation rate compared with that in solid tumors. Recurrent mutations occur in genes related to DNA methylation, chromatin, histone methylation, myeloid transcription factors, signaling, adhesion, cohesin complex, and the spliceosome. Their pattern of mutual exclusivity and cooperation among mutated genes suggests that these genes have a strong biological relationship. In addition, we identified mutations in previously unappreciated genes such as MLL3, NSD1, FAT1, FAT4, and IDH3B. Mutations in 9 genes were observed in the relapse-specific phase. DNMT3A mutations are the most stable mutations, and this DNMT3A-transformed clone can be present even in morphologic complete remissions. Of note, all AML matched trio samples shared at least 1 genomic alteration at diagnosis and relapse, suggesting common ancestral clones. Two types of clonal evolution occur at relapse: either the founder clone recurs or a subclone of the founder clone escapes from induction chemotherapy and expands at relapse by acquiring new mutations. Relapse-specific mutations displayed an increase in transversions. Functional assays demonstrated that both MLL3 and FAT1 exert tumor-suppressor activity in the FLT3-ITD subtype. An inhibitor of XPO1 synergized with standard AML induction chemotherapy to inhibit FLT3-ITD growth. This study clearly shows that FLT3-ITD AML requires additional driver genetic alterations in addition to FLT3-ITD alone.

Quantitative detection of DNMT3A R882H mutation in acute myeloid leukemia.

BACKGROUND: DNMT3A mutations represent one of the most frequent gene alterations detectable in acute myeloid leukemia (AML) with normal karyotype. Although various recurrent somatic mutations of DNMT3A have been described, the most common mutation is located at R882 in the methyltransferase domain of the gene. Because of their prognostic significance and high stability during disease evolution, DNMT3A mutations might represent highly informative biomarkers for prognosis and outcome of disease. METHODS: We describe an allele-specific PCR with a Blocking reagent for the quantitative detection of DNMT3A R882H mutation providing the possibility to analyze the quantitative amount of mutation during the course of disease. Next, we analyzed 62 follow-up samples from 6 AML patients after therapy and allogeneic stem cell transplantation (alloSCT). RESULTS: We developed an ASB-PCR assay for quantitative analysis of R882H DNMT3A mutation. After optimization of blocker concentration, a R882H-positive plasmid was constructed to enhance the accuracy of the sensitivity of quantitative detection. The assay displayed a high efficiency and sensitivity up to 10(-3). The reproducibility of assay analyzed using follow-up samples showed the standard deviation less than 3.1 %. This assay displayed a complete concordance with sequencing and endonuclease restriction analysis. We have found persistence of DNMT3A R882H mutations in complete remission (CR) after standard cytoreduction therapy that could be indicating presence of DNMT3A mutation in early pre-leukemic stem cells that resist chemotherapy. The loss of correlation between NPM1 and DNMT3A in CR could be associated with evolution of pre-leukemic and leukemic clones. In patients with CR with complete donor chimerism after alloSCT, we have found no DNMT3A R882H. In relapsed patients, all samples showed an increasing of both NPM1 and DNMT3A mutated alleles. This suggests at least in part the presence of NPM1 and DNMT3A mutations in the same cell clone. CONCLUSION: We developed a rapid and reliable method for quantitative detection of DNMT3A R882H mutations in AML patients. Quantitative detection of DNMT3A R882H mutations at different time points of AML disease enables screening of follow-up samples. This could provide additional information about the role of DNMT3A mutations in development and progression of AML.

Multiple myeloma cells alter the senescence phenotype of bone marrow mesenchymal stromal cells under participation of the DLK1-DIO3 genomic region.

BACKGROUND: Alterations and senescence in bone marrow mesenchymal stromal cells of multiple myeloma patients (MM-BMMSCs) have become an important research focus. However the role of senescence in the pathophysiology of MM is not clear. METHODS: Correlation between senescence, cell cycle and microRNA expression of MM-BMMSCs (n = 89) was analyzed. Gene expression analysis, copy number analysis and methylation specific PCR were performed by Real-Time PCR. Furthermore, cyclin E1, cyclin D1, p16 and p21 genes were analyzed at the protein level using ELISA. Cell cycle and senescence were analyzed by FACS. MiRNA transfection was performed with miR-485-5p inhibitor and mimic followed by downstream analysis of senescence and cell cycle characteristics of MM-BMMSCs. Results were analyzed by Mann-Whitney U test, Wilcoxon signed-rank test and paired t-test depending on the experimental set up. RESULTS: MM-BMMSCs displayed increased senescence associated ß-galactosidase activity (SA-ßGalA), cell cycle arrest in S phase and overexpression of microRNAs. The overexpressed microRNAs miR-485-5p and miR-519d are located on DLK1-DIO3 and C19MC, respectively. Analyses revealed copy number accumulation and hypomethylation of both clusters. KMS12-PE myeloma cells decreased SA-ßGalA and influenced cell cycle characteristics of MM-BMMSCs. MiR-485-5p was significantly decreased in co-cultured MM-BMMSCs in connection with an increased methylation of DLK1-DIO3. Modification of miR-485-5p levels using microRNA mimic or inhibitor altered senescence and cell cycle characteristics of MM-BMMSCs. CONCLUSIONS: Here, we show for the first time that MM-BMMSCs have aberrant methylation and copy number of the DLK1-DIO3 and C19MC genomic region. Furthermore, this is the first study pointing that multiple myeloma cells in vitro reduce both the senescence phenotype of MM-BMMSCs and the expression of miR-223 and miR-485-5p. Thus, it is questionable whether senescence of MM-BMMSCs plays a pathological role in active multiple myeloma or is more important when cell interaction with myeloma cells is inhibited. Furthermore, we found that MiR-485-5p, which is located on the DLK1-DIO3 cluster, seems to participate in the regulation of senescence status and cell cycle characteristics of MM-BMMSCs. Thus, further exploration of the microRNAs of DLK1-DIO3 could provide further insights into the origin of the senescence state and its reversal in MM-BMMSCs.

Association between low uric acid levels and acute graft-versus-host disease.

Endogenous danger signals are increasingly recognized in the pathogenesis of graft-versus-host disease (GVHD). Uric acid (UA) is a known danger signal and is released from injured cells during conditioning for allogeneic hematopoietic stem cell transplantation (HSCT), but its role in GVHD is unclear. Here, we retrospectively analyze 228 consecutive patients with malignant diseases undergoing HSCT from 10/10-HLA-matched donors. Low UA levels at the time of HSCT (day 0) were significantly associated with acute GVHD grades II-IV in univariate (HR 0.836, p = 0.0072) and multivariate analyses (HR 0.815, p = 0.0047). There was no significant association between UA levels and overall survival, non-relapse mortality, and relapse. This is the first report demonstrating a negative association between UA levels and acute GVHD. Due to the easy assessment and established pharmaceutical modification of UA, our findings are potentially clinically relevant. Confirmation in independent cohorts and further investigations into underlying mechanisms, such as reduced antioxidative capacity in hypouricemia, are warranted.

Some aspects of allogeneic stem cell transplantation in patients with myelodysplastic syndrome: advances and controversy.

Myelodysplastic syndrome (MDS) is a heterogeneous group of myeloid disorders. MDS remains a disease of elderly patients; moreover, the incidence of high risk MDS is proportionally greater in elderly patients, with increased frequency of secondary acute myeloid leukemia, as well as adverse cytogenetic abnormalities. Allogeneic stem cell transplantation is a therapeutic approach with known curative potential for patients with MDS that allows the achievement of long-term disease control. Numerous controversies still exist regarding transplantation in MDS: timing of transplantation, disease status at transplantation and comorbidity, conditioning intensity, pretransplant therapy, and stem cell source. Various transplant modalities of different intensities and alternative donor sources are now in use. Current advances in transplant technology are allowing the consideration of older patients. This should result in a greater number of older patients benefiting from this potentially curative treatment modality. Despite advances in transplantation technology, there is still considerable morbidity and mortality associated with this approach. Nevertheless, with the introduction of reduced-intensity conditioning and thereby reduced early mortality, transplant numbers in MDS patients have significantly increased. Moreover, recent new developments with innovative drugs, including hypomethylating agents, have extended the therapeutic alternatives for MDS patients. Hypomethylating agents allow the delay of allogeneic stem cell transplantation by serving as an effective and well-tolerated means to reduce disease burden.

Single-Nucleotide Polymorphisms Within the Thrombomodulin Gene (THBD) Predict Mortality in Patients With Graft-Versus-Host Disease.

PURPOSE: Steroid-refractory graft-versus-host disease (GVHD) is a major and often fatal complication after allogeneic stem-cell transplantation (alloSCT). Although the pathophysiology of steroid refractoriness is not fully understood, evidence is accumulating that endothelial cell stress is involved, and endothelial thrombomodulin (THBD) plays a role in this process. Here we assess whether single-nucleotide polymorphisms (SNPs) within the THBD gene predict outcome after alloSCT. PATIENTS AND METHODS: Seven SNPs within the THBD gene were studied (rs1962, rs1042579, rs1042580, rs3176123, rs3176124, rs3176126, and rs3176134) in a training cohort of 306 patients. The relevant genotypes were then validated in an independent cohort (n = 321). RESULTS: In the training cohort, an increased risk of nonrelapse mortality (NRM) was associated with three of seven SNPs tested: rs1962, rs1042579 (in linkage disequilibrium with rs3176123), and rs1042580. When patients were divided into risk groups (one v no high-risk SNP), a strong correlation with NRM was observed (hazard ratio [HR], 2.31; 95% CI, 1.36 to 3.95; P = .002). More specifically, NRM was predicted by THBD SNPs in patients who later developed GVHD (HR, 3.03; 95% CI, 1.61 to 5.68; P < .001) but not in patients without GVHD. In contrast, THBD SNPs did not predict incidence of acute GVHD. Multivariable analyses adjusting for clinical variables confirmed the independent effect of THBD SNPs on NRM. All findings could be reproduced in the validation cohort. CONCLUSION: THBD SNPs predict mortality of manifest GVHD but not the risk of acquiring GVHD, supporting the hypothesis that endothelial vulnerability contributes to GVHD refractoriness.

Comparative examination of various PCR-based methods for DNMT3A and IDH1/2 mutations identification in acute myeloid leukemia.

BACKGROUND: Mutations in epigenetic modifiers were reported in patients with acute myeloid leukaemia (AML) including mutations in DNA methyltransferase 3A gene (DNMT3A) in 20%-30% patients and mutations in isocitrate dehydrogenase 1/2 gene (IDH1/2) in 5%-15% patients. Novel studies have shown that mutations in DNMT3A and IDH1/2 influence prognosis, indicating an increasing need to detect these mutations during routine laboratory analysis. DNA sequencing for the identification of these mutations is time-consuming and cost-intensive. This study aimed to establish rapid screening tests to identify mutations in DNMT3A and IDH1/2 that could be applied in routine laboratory procedures and that could influence initial patient management. METHODS: In this study we developed an endonuclease restriction method to identify the most common DNMT3A mutation (R882H) and an amplification-refractory mutation system (ARMS) to analyse IDH2 R140Q mutations. Furthermore, we compared these methods with HRM analysis and evaluated the latter for the detection of IDH1 mutations. RESULTS: Of 230 samples from patients with AML 30 (13%) samples had DNMT3A mutations, 16 (7%) samples had IDH2 R140Q mutations and 36 (16%) samples had IDH1 mutations. Sensitivity assays performed using serial dilutions of mutated DNA showed that ARMS analysis had a sensitivity of 4.5%, endonuclease restriction had a sensitivity of 0.05% and HRM analysis had a sensitivity of 5.9%-7.8% for detecting different mutations. HRM analysis was the best screening method to determine the heterogeneity of IDH1 mutations. Furthermore, for the identification of mutations in IDH2 and DNMT3A, endonuclease restriction and ARMS methods showed a perfect concordance (100%) with Sanger sequencing while HRM analysis showed a near-perfect concordance (approximately 98%). CONCLUSION: Our study suggested that all the developed methods were rapid, specific and easy to use and interpret. HRM analysis is the most timesaving and cost-efficient method to rapidly screen all the 3 genes at diagnosis in samples obtained from patients with AML. Endonuclease restriction and ARMS assays can be used separately or in combination with HRM analysis to obtain more reliable results. We propose that early screening of mutations in patients with AML having normal karyotype could facilitate risk stratification and improve treatment options.

Bone marrow T-cell infiltration during acute GVHD is associated with delayed B-cell recovery and function after HSCT.

B-cell immune dysfunction contributes to the risk of severe infections after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Delayed B-cell regeneration is found in patients with systemic graft-versus-host disease (GVHD) and is often accompanied by bone marrow (BM) suppression. Little is known about human BM GVHD. We analyzed the reconstitution kinetics of B-cell subsets in adult leukemic patients within 6 months after allo-HSCT. B-cell deficiency already existed before transplant and was aggravated after transplant. Onset of B-cell reconstitution characterized by transitional B-cell recovery occurred either early (months 2-3) or late (from month 6 on) and correlated highly positively with reverse transcription-polymerase chain reaction quantified numbers of κ-deleting recombination excision circles (KRECs). Delayed recovery was associated with systemic acute GVHD and full-intensity conditioning therapy. Histological analysis of BM trephines revealed increased T-cell infiltration in late recovering patients, which was associated with reduced numbers of osteoblasts. Functionally, late recovering patients displayed less pneumococcal polysaccharide-specific immunoglobin M-producing B cells on ex vivo B-cell activation than early recovering patients. Our results provide evidence for acute BM GVHD in allo-HSCT patients with infiltrating donor T cells and osteoblast destruction. This is associated with delayed B-cell reconstitution and impaired antibody response. Herein, KREC appears suitable to monitor BM B-cell output after transplant.

Bone marrow stromal cells in the pathogenesis of acute myeloid leukemia.

Acute myeloid leukemia (AML) is clonal disorder affecting pluripotent stem cell and characterized by ineffective hematopoiesis. Genetic abnormalities in a progenitor cells is thought to lead to uncontrolled growth of leukemia cells. In addition, in the last years, it has been clearly recognized that the hematopoietic microenvironment (HM) plays an important role in the pathogenesis of AML. The HM can regulate hematopoiesis by interacting directly with HC and/or by secreting regulatory molecules that exert a positive or negative influence on the growth of HC. Stromal elements are important in the homing of immature HC or hematopoietic stem cells. Several studies propose that important quantitative and functional alterations are present in the BMSC of AML patients. AML may arise in the setting of an abnormal HM, resulting in the generation of multiple populations with varying initiation event. Dysfunction of HM may contribute to leukemia by supplying abundant growth factors that promote proliferation and/or inhibit apoptosis. Recent discoveries utilizing mouse models showed that genetic alteration in cells of HM can induce AML.

JAK2 V617F allele burden quantified by real time quantitative polymerase chain reaction and competitive polymerase chain reaction in patients with chronic myeloproliferative neoplasia.

Assessing the clinical significance of JAK2 V617F mutant allele burden is complicated by a myriad of techniques reported to detect and quantify the mutation. As a consequence, the level of sensitivity and how the data is reported vary. Harmonization of well-defined molecular studies would permit evaluation of the clinical significance of measuring allele burden and rapid determination of the efficacy of novel agents for the treatment of chronic myeloproliferative neoplasia via multicenter clinical trials, at the subclinical level. Here we report a comparison between the widely available TaqMan quantitative real time polymerase chain reaction (Q-PCR) and competitive PCR (C-PCR) assays. We found that the tumor load was invariably greater when measured by C-PCR compared to that recorded by Q-PCR. Furthermore, none of the samples converted from undetectable to detectable when the enriched granulocyte (GR) fraction was tested. While a difference in the V617F allele levels was detected between GR fraction and whole blood, this was not statistically significant.